Characterization of plant growth-promoting rhizobacteria using capillary isoelectric focusing with whole column imaging detection

Journal of Chromatography A, 1140 (2007) 213–218

Characterization of plant growth-promoting rhizobacteria using capillary isoelectric focusing with whole column imaging detection Zhen Liu a,∗ , Shan Shan Wu b , Janusz Pawliszyn c a

Department of Chemistry, Nanjing University, Nanjing 210093, China Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada c Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada b

Received 10 October 2006; received in revised form 27 November 2006; accepted 29 November 2006 Available online 12 December 2006

Abstract Capillary isoelectric focusing (cIEF) can be a useful tool for the characterization and identification of microbes. Based on the whole column imaging detection (WCID) technique and using plant growth-promoting rhizobacteria (PGPR) as test microbes, we present a two-level cIEF characterization method for the characterization and identification of bacteria. Intact bacteria were first characterized according to their apparent isoelectric points measured by cIEF-WCID and then lysed bacteria were further characterized by cIEF profiling of the intracellular proteins. Cellular clustering was found to be the main experimental barrier for the characterization of intact bacteria. The addition of sodium chloride (100 mM) to the sample mixture was found to be an effective way to reduce clustering. Due to the high efficiency and high resolution of cIEF-WCID, characterization of bacteria according to their intracellular proteins can be implemented simply and quickly without optimization of the experimental conditions. To improve the detection sensitivity with laser induced fluorescence (LIF)-WCID, the possibility to label bacteria with a non-covalent fluorescent dye, NanoOrange, was explored. © 2006 Elsevier B.V. All rights reserved. Keywords: Bacteria; Capillary isoelectric focusing; Characterization; Laser-induced fluorescence; Whole column imaging detection

1. Introduction The characterization and identification of microbes such as bacteria is of great importance in many fields, including bioscience research, medical diagnosis and the food industry. Currently, a variety of conventional methods exist for the characterization and identification of microbes, for example, differential staining, flow cytometry, phage typing, protein analysis, and the comparison of nucleic acid (rRNA or rDNA) sequences. However, many of these approaches are timeconsuming because of the number of the experimental steps involved in the process. Instrumental techniques for the analysis and characterization of microbes are becoming more common. Although these new approaches will not replace the traditional methods in the immediate future, they can be useful alternatives or complements. In particular, capillary electrophoresis (CE) based methods seem to be very promising.

∗

Corresponding author. Tel.: +86 25 8368 5639; fax: +86 25 8368 5639. E-mail address: [email protected] (Z. Liu).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.093

Since the initial development of electrophoresis, it has been known that colloidal particles migrate under the influence of an electric field [1]. Later, Hjert´en [2,3] demonstrated that microbes can be separated by zone electrophoresis. Meanwhile, isoelectric focusing (IEF) was employed to evaluate the surface charge characteristics of bacteria [4–6]. Soon after the invention of CE, CE-based approaches were used to separate microorganisms [7–12]. Particularly, since the work of Armstrong et al. [13], who reported the successful separation of microbes by CE in the manner of molecules, CE methods for the separation, characterization and identification of microbes became more popular [14–29]. Two separation modes, capillary zone electrophoresis (CZE) [11,13,14,26–29] and capillary isoelectric focusing (cIEF) [10,13,15,22] have been employed. Despite the same separation mechanism, the behavior of microbes in CE is more complicated than that of molecules. First, microbial clustering is often encountered [16,25,26,30,31]; a single strain of microbe can exhibit non-reproducible peak clusters, which makes the characterization and identification impossible. Although brief sonication of the sample is a simple way to prevent cellular clustering, it does not effectively disperse some microbes that are

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strongly associated [16]. Second, apparent ultrahigh efficiencies have been observed for CZE separations of microbes (∼106 to 1010 theoretical plates/m) [13,20,21]. The ultrahigh efficiencies are beneficial to the separation and detection sensitivity, though the mechanism has not been fully understood. For the purpose of characterization and identification of microbes, cIEF seems to be a more promising technique. Since the electrophoretic mobility of microbes is dependent on experimental conditions while the isoelectric point (pI) value is usually independent, some investigators [10,15] have already suggested that the use of the pI value is a better method to establish identification of microorganisms in a sample. cIEF with the whole-column imaging detection (WCID) [32–36] is a unique technique, in which a short separation capillary (usually several centimeters long) is used and the focused sample bands in the whole column range are detected by imaging detection with a CCD camera. By using WCID, the mobilization step necessary for conventional cIEF can be avoided, offering significant advantages such as maintained high efficiency and resolution, reduced separation time and easy method development. cIEF-WCID has found applications in the separation and characterization of small molecules [37], peptides [38,39,46], proteins [38–40], antibodies [39,41] and viruses [41–43]. It has been demonstrated as a useful tool for the study of reactions and interactions of proteins [44] and for quick two-dimensional characterization of proteins [45]. Based on the cIEF-WCID technique, we present a two-level characterization method for the characterization and identification of bacteria with plant growth-promoting rhizobacteria (PGPR) as test bacteria. Intact bacteria are first characterized by cIEF-WCID according to their apparent pI’s. Then the bacteria are lysed by sonication, and the lysed bacteria are further characterized by cIEF profiling of the intracellular proteins. In order to improve the detection sensitivity, we attempted to characterize the bacteria using cIEF-laser induced fluorescence (LIF)-WCID through non-covalent labeling with NanoOrange.

2. Experimental 2.1. Instrument The cIEF-UV-WCID experiments were performed on an iCE 280 instrument (Convergent Bioscience, Toronto, Canada) with UV absorption detection at 280 nm. Cartridges of 5 cm × 100 ␮m i.d. internally fluorocarbon-coated fused-silica capillary were purchased from Convergent Bioscience. The instrument set-up for cIEF-LIF-WCID was built in-house as described previously [43]. The excitation laser beam (488 nm) was produced by an air-cooled argon ion laser and the detection filter was a longpass filter cut-off at 530 nm. Cartridges with separation channels of a 5 cm long (effective length) and 150 ␮m i.d. Teflon AF 2400 capillary were used. Cell disruption was performed in a Branson Sonifier 200 cell disruptor (Branson Ultrasonic, Danbury, CT, USA).

2.2. Reagents and materials Four strains of the plant growth-promoting rhizobacteria were used: UW3 (Pseudomonas putida) and MG3 (Pseudomonas fluorescens), UW4 (Enterobacter cloacae) and EP3 (Alkaligene xylosoxydans). The strains were cultured in 100 mL of tryptic soy broth (TSB) medium and proliferated on a shaker at 200 rpm at room temperature for 24 h. The cell numbers of the cultured bacterial solutions were determined by the spectrometric method. Optical density (OD) readings at 600 nm were obtained from a UV-2101 spectrophotometer (Shimadzu, Columbia, MD, USA). The OD readings of the bacteria were compared to a standard curve constructed by dilution plate count procedure. The bacterial cell densities were determined to be 1.6, 2.2, 1.4, 5.1 × 109 cells/mL for UW3, EP3, UW4 and MG3, respectively. Pharmalytes (pH 3–10, 40% in concentration) and polyvinylpyrrolidone (PVP, average molecular weight about 360,000 and intrinsic viscosity 80–100 K) were purchased from Sigma (St. Louis, MO, USA). The pI markers were kindly donated by Dr. Karel Slais (Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic). The anolyte and catholyte were 100 mM phosphoric acid and 100 mM sodium hydroxide, respectively. Water was purified with an ultra-pure water system (Barnstead/Thermolyne, Dubuque, IA, USA), and was used to prepare all solutions. The NanoOrange protein quantitation kit was purchased from Invitrogen (formerly Molecular Probes, Eugene, OR, USA). 2.3. Methods Before separation, the separation capillary was conditioned with water and a 0.5% (w/v) PVP aqueous solution for 20 min each. The sample was injected to fill the whole separation capillary. For the cIEF-UV-WCID experiments, the focusing was initiated by applying 500 V for 2 min and then the voltage was increased to 3 kV. Imaging was taken automatically at desired intervals. For the cIEF-LIF-WCID experiments, the focusing was initiated by applying 500 V for 2 min and then the voltage was ramped to 2.5 kV. Imaging was taken manually at desired times. When the focusing current reached a constant residual current and two consecutive focusing profiles were the same, the focusing was completed. For the study of intact bacterial samples, the samples were prepared by mixing 1% bacterial solution, 2% Pharmalytes (pH 3–10) and 0.5% PVP, without or with certain additives such as glycerol or sodium chloride. For the study of lysed bacteria, the bacterial cells were disrupted by sonication in an ice-bath for a total of 12 min with a 1-min break per 2-min of sonication (energy output set at 40 W). An aliquot of the disrupted bacteria samples was prepared into a final sample solution that contained 5% disrupted bacterial solution, 2% Pharmalytes (pH 3–10) and 0.5% PVP. For non-covalent labeling, a NanoOrange working solution (1×) was prepared according to the vendor instructions. The labeling procedure was modified by incubating the solution at room temperature. An aliquot of the bacterial solution (10 ␮L) was mixed with 200 ␮L of 1× working solution, incubated at

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room temperature for 30 min, protected from light. The incubated solution was then prepared into 1 mL sample mixture containing 2% Pharmalytes (pH 3–10) and 0.5% PVP. 3. Results and discussion 3.1. Intact bacteria cIEF experiments of intact bacteria were first carried out under standard cIEF-WCID conditions (without the addition of any additives in the sample solution). The focusing time took 8 min. As shown in Fig. 1, UW3 exhibited one major peak and one minor peak, while the strains EP3, UW4 and MG3 exhibited multiple peaks. These multiple peaks were attributed to cellular clustering. Repeated experiments showed that the reproducibility was very poor due to cellular clustering. For example, even for UW3, which exhibited the least extent of clustering, the reproducibility was poor, with a relative standard deviation (RSD) value for peak position of 24.7% (n = 9). Therefore, it was determined that in order to improve the efficiency of this method, cellular clustering had to be suppressed or eliminated. Brief sonication of the sample solutions prior to injection was considered to be a possible method to eliminate cellular clustering, however, it was found to be ineffective for the bacteria investigated in this study. Another approach that was considered was to reduce the bacterial concentration in the sample solution. Unfortunately, cellular clustering still occurred even when the bacterial concentration was diluted by 10 times (∼106 cells/mL). Further dilution was not pursued because it was beyond the detection limit of the detector. Solubilizers such as glycerol have been used to prevent protein precipitation in cIEF for the separation of proteins with poor solubility [46]. Thus, the third approach tested in this study involved the addition of glycerol to the sample solution; unexpectedly, the addition of glycerol (20%, v/v) caused more severe clustering (data not shown). Finally, we turned to the addition of sodium chloride, because the work of Hork´a et al. [22] has showed that clustering could

Fig. 1. cIEF profiles of intact PGPRs. Samples: 1% bacteria containing 2% Pharmalytes (pH 3–10) and 0.5% PVP.

Fig. 2. EP3 cIEF profiles for six consecutive runs with the addition of sodium chloride in the sample. Samples: 1% EP3 containing 2% Pharmalytes (pH 3–10), 0.5% PVP and 100 mM NaCl; control: 1% TSB medium containing 2% Pharmalytes (pH 3–10), 0.5% PVP and 100 mM NaCl. The small peaks in the range of 0.6–1.2 cm and the broad “hump” in the range of 2.1–2.7 cm were due to the TSB culture medium that contained protein digests.

be avoided for bacterial samples prepared with a physiological saline solution and our previous work [41] has also found that the presence of salt in the sample solution is favorable for improving the peak shape and reproducibility of virus samples. EP3, which exhibited the most severe clustering, was used for the sodium chloride experiments. As shown in Fig. 2, the presence of sodium chloride (100 mM) greatly reduced cellular clustering. Although there were still several peaks in each run, it seems that each peak was due to a fixed aggregation number, since the peak positions were quite repeatable (RSD < 3.5%). In each run, the EP3 strain always exhibited a major peak and several smaller peaks. Because of its abundance, the major peak was chosen to calculate the pI value. As it is not clear at present whether the major peaks represent individual bacterial cells or cellular clusters, the pI measured is an apparent value for the bacterial cells. It has been found that the presence of salt in the sample mixture may result in a compacted pH gradient in cIEF [47]. The salt effect was rationalized with a mechanism similar to that of the blocking reagent, N,N,N ,N -tetramethylethylenediamine (TEMED) [47]. During the focusing process, salt is stripped out of the sample solution under the influence of an applied electric field. It migrates towards the two ends of the separation capillary and finally forms two zones and, therefore, squeezes the pH gradient inside the capillary. The phenomenon of pH gradient compression was clearly observed in Fig. 3. Moreover, it was observed that the addition of sodium chloride in the sample mixture apparently facilitated the focusing, reducing the focusing time from 8 min, in the absence of sodium chloride, to 5 min, in the presence of 100 mM sodium chloride. It is mainly due to reduced effective focusing length of the capillary. The usefulness of the pH gradient with the presence of sodium chloride was investigated. Calibration curves were established with seven low-molecular mass pI markers for the cases without and with

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Fig. 3. Comparison of the cIEF profiles of standard pI markers without (A) and with (B) the presence of sodium chloride in the sample. Samples: 0.5% pI markers containing 2% Pharmalytes (pH 3–10), 0.5% PVP without (A) and with (B) 100 mM NaCl.

Fig. 4. cIEF profiles of EP3, UW3 and UW4. Samples: 1% bacteria containing 2% Pharmalytes (pH 3–10), 0.5% PVP and 100 mM NaCl; control: 1% TSB medium containing 2% Pharmalytes (pH 3–10), 0.5% PVP and 100 mM NaCl. cIEF profile of EP3 is the same as Run #2 in Fig. 2. The small peaks in the range of 0.6–1.2 cm and the broad “hump” in the range of 2.1–2.7 cm were due to the TSB culture medium that contained protein digests.

the presence of sodium chloride, as given in Eqs. (1) and (2), respectively. pH = 3.11(±0.16) + 1.41(±0.06)X, r = 0.9951,

SD = 0.23,

n=7

(1)

pH = 1.27(±0.23) + 2.32(±0.10)X, r = 0.9956,

SD = 0.22,

n=7

(2)

where X is the peak position from the anode in centimeters. Clearly, the linearity of the pH gradient was observed to be the same, regardless of the addition of sodium chloride. Thus the pH gradient established with the presence of sodium chloride can be used for pI measurement. Fig. 4 illustrates the cIEF profiles of EP3, UW3 and UW4 with the presence of 100 mM sodium chloride in the sample mixture (cIEF profile of MG3 is not included because of a mistake during sample preparation). The pI values the major peaks of these bacterial strains were measured to be 4.71, 4.20 and 4.34, respectively. 3.2. Intracellular proteins Protein analysis is a useful way to characterize microbes. However, traditional gel electrophoresis for protein analysis is time-consuming and mass spectrometry-based methods require costly instruments. CE separations of lysed bacteria have been previously attempted [13,22]; however, the intracellular proteins were found to be much more difficult to separate than intact bacteria. Due to its high efficiency and high resolution, cIEF-WCID can be an effective approach for profiling intracellular proteins. The intracellular proteins were released from the bacterial cells by sonication disruption. The sonication time was set long enough (12 min, as compared with 50 s for extremely hardy cells [48]) to ensure the bacterial cells were completely broken down. The disrupted bacterial solutions were directly prepared into samples for analysis, without

further treatment. Peaks observed in cIEF-WCID were considered to represent primarily intracellular proteins or peptides of the bacteria, because of several reasons: (1) charged intracellular components such as nucleic acids will be electrokinetically pumped out of the separation column under the applied electric field; (2) focusing of amino acids usually takes much longer time (>30 min) and resulting peaks are usually very broad (due to the flat titration curve of amino acids); (3) other non-charged and amphoteric intracellular components that may still remain in the separation column during the focusing, such as phospholipids (the major components of cell membranes), are not detectable at the chosen detection wavelength of 280 nm. In this study, the intracellular proteins of the four PGPR strains were profiled by cIEF-WCID under standard conditions without the addition of sodium chloride. The focusing was completed in 8 min. Without the presence of sodium chloride in the sample mixture, the reproducibility of peak position was better (RSD < 2.0%). As shown in Fig. 5, the intracellular proteins of the bacteria investigated were separated well, and each bacterial strain exhibited a characteristic cIEF protein profile. The cIEF profiles can be divided into three main regions: (1) background region, in the range of 2.5–3.2 cm, peaks in this region were due to protein digests of the TSB medium; (2) common region, in the range of 1.5–2.0 cm, there was only one peak, which is most likely representative of a common intracellular protein in the four strains; and (3) fingerprint region, in the range of 0–1.5 cm, the peaks in this region are characteristic for certain strains. Thus, it can be seen that the common region can be used to identify if a bacterium belongs to a specific species and the fingerprint region can be used to determine if a bacterium belongs to a specific strain. However, to identify multiple bacteria in a mixture is difficult with the method if the fingerprint regions of different strains overlap each other.

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Fig. 5. cIEF profiles of intracellular proteins of the PGPRs. Samples: 5% disrupted bacteria containing 2% Pharmalytes (pH 3–10) and 0.5% PVP. Control: 1% TSB medium containing 2% Pharmalytes (pH 3–10) and 0.5% PVP. (I) Background region; (II) common region; (III) fingerprint region.

3.3. Non-covalent labeling with NanoOrange Non-covalent fluorescent labeling is an important means for fluorescence detection. NanoOrange is a typical non-covalent labeling dye for protein analysis. It is weakly fluorescent in its unbound state; however, it emits strong fluorescence once bound to proteins. The binding is mainly through hydrophobic interactions. Labeling with NanoOrange permits accurate detection of proteins in solution ranging from 10 ng/mL to 10 ␮g/mL [49,50]. When bound to proteins, this dye has a broad excitation peak centered at ∼470 nm and a broad emission peak at ∼570 nm. NanoOrange also has been used to label antibodies and viruses [41,43]. However, to our best knowledge, there have been no reports of non-covalent fluorescent labeling of cells to date. Because the cell surface of bacteria contains structural proteins, which may have hydrophobic moieties, non-covalent fluorescent labeling of microbes seems to be a feasible approach and was tested in this study. As shown in Fig. 6, the four bacterial strains were effectively labeled with NanoOrange. The strain UW4 exhibited two major peaks, and the reason for this is not clear at present. Compared with un-labeled bacteria, the non-covalent labeling with NanoOrange offered a sensitivity enhancement of at least 40-fold. Moreover, the NanoOrange-labeled PGPRs exhibited dramatically reduced cellular clustering, as compared with un-labeled bacteria in the absence of sodium chloride. Unexpectedly, the peak width became much wider. The broadened peaks were most likely due to multi-labeling, which caused heterogeneity of the microbes. 3.4. Interactions influencing cellular clustering Based on the findings in this study, intermolecular interactions that influence cellular clustering can be briefly discussed. Bacterial cells have high specific surface area and exhibit high tendency of clustering, because of their very small sizes. The high tendency of clustering is resulted from not only

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Fig. 6. cIEF-LIF-WCID profiles of NanoOrange-labeled PGPRs. Samples: 1% bacteria containing 0.2× NanoOrange, 2% Pharmalytes (pH 3–10) and 0.5% PVP.

the high specific surface area but also intercellular interactions. Cell membranes consist of lipids and associated proteins. These molecules may involve in hydrogen bond, electrostatic and hydrophobic interactions, depending on the conditions of surrounding solution. The addition of glycerol caused more severe clustering. This effect is likely due to the fact that glycerol has three hydroxyl groups so that it can link cells through hydrogen bond. The addition of sodium chloride greatly reduced cellular clustering. This effect is probably because the presence of sodium chloride decreases electrostatic attractions between cells. The labeling with NanoOrange diminished cellular clustering. As NanoOrange interacts with proteins mainly through hydrophobic interaction, the reduced cellular clustering observed seems related to the decreased hydrophobic surface area of the bacterial cells due to the occupation of surface sites by NanoOrange. 4. Conclusion On the basis of cIEF-WCID, we have presented a two-level bacterial characterization method in this study. The first level is to directly characterize intact bacteria, offering apparent pI values for bacterial cells. Cellular clustering is the key aspect that needs to be considered at this level. The addition of sodium chloride to the sample mixture was found to be an effective way to reduce clustering. The second level is to profile the intracellular proteins of bacterial samples disrupted by sonication. Due to the high efficiency and high resolution of cIEF-WCID, this level of characterization can be implemented readily under standard cIEF-WCID conditions without further optimization. The cIEF profiles of intracellular proteins can be useful for identifying what species and what strain a sample bacterium belongs to once a database for known species and strains have been established. Compared with the conventional methods for characterization of microbes, the most significant advantages of this method include speed and ease in method development.

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Acknowledgment The authors thank Dr. Karel Slais at the Institute of Analytical Chemistry, the Academy of Sciences of the Czech Republic, for donation of the pI markers. Z.L. acknowledges National Natural Science Foundation of China for financial support (Grant No. 20521503). S.S.W. thanks Dr. Bernard R. Glick and Dr. Bruce M. Greenberg (Department of Biology, University of Waterloo) for donation of the bacteria and providing the equipment for bacteria culturing. References [1] A. Tiselius, Nova Acta Regiae Soc. Sci. Ups., Ser. IV 7 (1930) 1. [2] S. Hjert´en, Free Zone Electrophoresis, Almquist and Wiksells, Uppsala, 1967. [3] H. Bloemendal (Ed.), Cell Separation Methods, Elsevier, Amsterdam, 1977. [4] O. Stendahl, L. Edebo, K.E. Magnusson, C. Tagesson, S. Hjert´en, Acta Pathol. Microbiol. Scand. 85 (1977) 334. [5] R.W. Longton, J.S. Cole III, QuinnF P.E., Arch. Oral Biol. 20 (1975) 103. [6] E. Jaspers, J. Overmann, Appl. Environ. Microbiol. 63 (1997) 3176. [7] S. Hjert´en, K. Elenbring, F. Kil´ar, J.-L. Liao, A.J.C. Chen, C.J. Siebert, M.-D. Zhu, J. Chromatogr. 403 (1987) 47. [8] A. Zhu, Y. Chen, J. Chromatogr. 470 (1989) 251. [9] R.C. Ebersole, R.M. McCormick, Bio/Technology 11 (1993) 1278. [10] U. Schnabel, F. Groiss, D. Blaas, E. Kenndler, Anal. Chem. 68 (1996) 4300. [11] V.M. Okun, B. Ronacher, D. Blaas, E. Kenndler, Anal. Chem. 71 (1999) 2028. [12] A. Pfetsch, T. Welsch, Fresenius J. Anal. Chem. 359 (1997) 198. [13] D.W. Armstrong, G. Schulte, J.M. Schneiderheinze, D.J. Westenberg, Anal. Chem. 71 (1999) 5465. [14] D.W. Armstrong, J.M. Schneiderheinze, Anal. Chem. 72 (2000) 4474. [15] Y. Shen, S.J. Berger, R.D. Smith, Anal. Chem. 72 (2000) 4603. [16] J.M. Schneiderheinze, D.W. Armstrong, G. Schulte, D.J. Westenberg, FEMS Microbiol. Lett. 189 (2000) 39. [17] D.W. Armstrong, L. He, Anal. Chem. 73 (2001) 4551. [18] D.W. Armstrong, J.M. Schneiderheinze, J.P. Kullman, L. He, FEMS Microbiol. Lett. 194 (2001) 33. [19] K. Yamada, M. Torimura, S. Kurata, Y. Kamagata, T. Kanagawa, K. Kano, T. Ikeda, T. Yokomaku, R. Kurane, Electrophoresis 22 (2001) 3413.

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